Method for non-radioactive detection of membrane-bonded nucleic acids and test kit

ABSTRACT

The present invention relates to a novel method for non-radioactive detection of membrane-bonded nucleic acids, including nucleic acids that, for instance, contain single nucleotide polymorpbisms (SNP&#39;s), DNA arrays (cosmide, yeast, artificial chromosones (YAC&#39;s), bacterial artificial chromosones (BAC&#39;s), cDNA&#39;s, PCR fragments, oligonucleotides), RNA arrays and all nucleic acid fragments that are tranfered from gels (agarose or PAA) to membranes, including genomic DNA/plasmid DNA fragments (southern) and mRNA&#39;s (northern). The invention also relates to a test kit to carry out said method.

This application is the national phase under 35 U.S.C. § 371 of PCTInternational Application No. PCT/DE99/01066 which has an Internationalfiling date of May 3, 1999, which designated the United States ofAmerica.

DESCRIPTION

The present invention relates to a novel method for non-radioactivedetection of membrane-bonded nucleic acids, including nucleic acidsthat, for instance, contain single nucleotide polymorphisms (SNPs), DNAarrays (cosmids, yeast artificial chromosomes (YAC's), bacterialartificial chromosomes (BAC's), cDNA's, PCR fragments,oligonucleotides), RNA arrays and all nucleic acid fragments that aretransferred from gels (agarose or polyacrylamide (PAA) gels) tomembranes, including genomic DNA/plasmid DNA fragments (Southern) andmRNAs (Northern) and a test kit to apply the method.

Arrays are individual nucleic acids preferably arranged to a defineddegree on a membrane in rasters.

The identification of exchanges of individual bases within DNA (SNP's)is of great importance to investigating the causes of complex diseases,e. g. hypertension, and for diagnostic purposes (Lander, E. S. (1996):Science 274, 536-539, Collins, F. S. et al. (1997) Science 278,1580-1581, Marshall E. (1997): Science 277, 1752-1753). Such an exchangeis may be detected by sequencing the interesting regions.

Traditionally the sequence is determined by the enzymaticchain-terminating method where the sequencing products are marked by theincorporation of radioactively or non-radioactively labeled nucleotides.Following gel electrophoretic separation the sequence may be determinedby evaluation of the sequence gels. Direct labelling of the sequencingproducts, however, has the disadvantage that only one single DNAfragment may be analyzed per sequencing batch.

Multiplex technology (see EP-A 0 303 459) overcomes this disadvantage bysimultaneously sequencing a few DNA molecules. The resulting mixtures ofsequencing products formed are gel electrophoretically separated,transferred to a nylon membrane and fixed there. By hybridizing thismembrane with a probe which is specific for a single DNA fragment thesequences of a few DNA fragments may be successively read using the samemembrane. However, a big disadvantage of this method is that thedetection of various DNA fragments is effected by using radioactivelylabelled probes. That is why automation of the whole process to furtherincrease the flow rate will not be feasible.

Furthermore, a non-radioactive detection method is known which isdescribed by Richterich and Church (Richterich, R., Church, G. M (1993)Methods Enzymol 218: 187-222), however showing the disadvantages to benot suitable for successive hybridizations and usually hybridizationsmay not be carried out exclusively at room temperature.

The objective of the present invention is to establish an easy toperform non-radioactive method to be applied which maintains or evenimproves, on the one hand, the sensitivity of radioactive methods and,on the other hand, allows an immediate, direct successive detection.

The invention is implemented according to the claims. Surprisingly, thistask may be solved by means of a method which may be applied in allstages at room temperature. The method according to the invention issuited for a non-radioactive detection of membrane-bound nucleic acids(A) by hybridizing NAs bound to a membrane which are obtained by meansof methods known per se with a special hybridization buffer containingTris HCl, Tris base, NaCl, Triton X-100, SDS and a blocking reagent andsubsequently detecting the bound NA. The invention does not include thedetection of sequencing products according to the multiplex method inaccordance with EP 303 459 (G. Church).

Preferably membrane-bound DNA is detected.

The method is preferably characterized by the following steps:

1. Prehybridization of a membrane containing a DNA according to a methodknown per se, preferably in a dish, with the aid of a specialhybridization buffer,

2. hybridization of the membrane by means of a 5′biotinylated probe inthe same buffer,

3. repeated washing of the membrane with buffer I containing PBS(Na₂HPO4, NaH₂PO₄, NaCl, pH approx. 7.3), SDS and blocking reagent,

4. incubation of the membrane with streptavidin alkaline phosphataseconjugate in buffer I,

5. one-time washing of the membrane with buffer I, thereupon repeatedwashing with buffer II, containing PBS and SDS and repeatedequilibration at pH 9.75 with buffer III containing Tris-HCI anddiethanol amine,

6. after equilibration, transfer of the membrane into a substratesolution consisting of CDPstar® (disodium2-chloro-5-(4-methoxyspiro{1,2-dioxetane-3,2′-(5-chloro)-tricyclo[3.3.1.1^(3,7)]decan}-4-yl)-1-phenylphosphate, Tropix® and buffer III and, if necessary, agitation of themembrane to reach a uniform distribution of the substrate solution,

7. after short drainage, fixing of the membrane, e.g. onto a piece ofplastic, and covering, preferably with a transparent foil,

8. exposing, preferably by means of an X-ray film or a CCD camera,

9. after exposure returning of the membrane and repeated stripping witha preheated buffer IV containing EDTA and SDS,

10. one-time washing with buffer V containing Tris-HCl and NaCl.

The evaluation is performed according to established methods such ase.g. the ‘skilled pattern analysis’ principle.

The most important step in the above method is hybridization with aspecial hybridization buffer, thereby allowing repeated subsequenthybridization and a sensitivity equivalent to or better than radioactivedetection is obtained. Thus, the method according to the invention forthe non-radioactive detection of nucleic acids containing e.g. ‘singlenucleotide polymorphisms’ (SNP's) is to be regarded as a greatimprovement. In addition, the use of radioactivity which is hazardous tohealth may be avoided. Apart from that, by summarizing otherwisetime-consuming intermediate steps and optimizing the compositions ofsolutions and the necessary washing parameters constant results may beachieved allowing to provide the basis for the future development ofautomated detection.

The test kit according to the present invention for the non-radioactivedetection of nucleic acids containing e.g. ‘single nucleotidepolymorphisms’ (SNPs) contains the following solutions:

Hybridization buffer: 5 l

500 ml 10×TNT

1250 ml of 20% SDS

10 g of blocking reagent (Boheringer Mannheim, DIG kit)

3250 ml of H₂O (twice distilled)

10×PBS: 5 l

516.2 g of Na₂HPO₄

132.6 g of NaH₂PO₄

198.7 g of NaCl

H₂O to 5 l pH: approx. 7.3

Buffer I: 5 l

10 g of blocking reagent

250 ml 10×PBS

125 ml of 20% SDS

4625 ml of H₂O (twice distilled), to be heated for 8 min. in a microwaveoven, to be stirred until the blocking reagent is dissolved.

Buffer II: 5 l

250 ml 10×PBS

125 ml of 20% SDS

4625 ml of H₂O (twice distilled)

10×buffer III: 2 l

64.4 g of Tris HCl

1770 ml of H₂O (twice distilled)

210.4 g of diethanol amine pH: 9.75

Buffer IV: 5 l

4700 ml of H₂O (twice distilled)

50 ml of 0.5 M EDTA, pH 8.0

250 ml of 20% SDS

Buffer V: I 1 l

920 ml of H₂O (twice distilled)

30 ml of 5 M NaCl

50 ml of 1 M TrisCl, pH 8.0

10×TNT: 1 l 44.4 g of Tris HCl

25.6 g of Tris base

73 g of NaCl

818 ml of H₂O ( )

100 ml of Triton X-100 pH: approx. 8.0

EXAMPLE

All steps are carried out at room temperature.

1. Membrane with the DNA side up in a dish with 180 ml hybridizationbuffer (28 mM Tris HCI/21 mM Tris base/125 mM NaCl/1% of Triton X-100/5%of SDS/0.2% of block reagent) to be prehybridized for 1 h.

2. Discard prehybridization solution. Centrifuge 5′ biotinylated probefor 5 min. at max. speed and add it immediately in a final concentrationof 10 pmol/ml to 100 ml of hybridization buffer (200 μM/5 μl). Hybridizemembrane for 1 h.

3. Remove hybridization solution completely. Wash membrane 5× for 5 min.with 120 ml of buffer I (29 mM Na₂HPO₄/8 mM NaH₂PO₄/34 mM NaCl/0.5% ofSDS, 0.2% of block reagent). Remove washing solution always completely.Make sure that big air bubbles will not be below the membrane.

4. Remove washing solution completely. Incubate membrane for 10 min.with 4 μl streptavidin alkaline phosphatase conjugate (alkalinephosphatase conjugate is obtained from Boehringer Ingelheim; centrifugeit for 5 min. at max. speed before adding it) in 100 ml buffer I.

5. Remove SvAP solution completely. Wash membrane 1× for 5 min. with 120ml of buffer I and subsequently 6× for 5 min. with 120 ml of buffer II(29 mM Na₂HPO₄/8 mM NaH₂PO₄/34 mM NaCl/0.5% of SDS). Equilibratesubsequently the membrane 4× for 5 min. with 120 ml of buffer III (20 mMTris HCI/100 mM diethanol amine/pH: 9.75).

6. In the mean time add 50 μl of CDPstar® (Tropix) to 10 ml of bufferIII (1:200) and mix it thoroughly. Subsequently add the substratesolution into the middle of the ‘CDPstar®only’ dish.

7. Take membrane from the dish after completing the last equilibrationstep and drain it briefly. Subsequently the membrane will be placed ontothe substrate solution with the DNA side down. By taking up and puttingdown the membrane repeatedly it is made sure that the substrate solutionwill be distributed uniformly below the membrane.

8. Drain the membrane briefly and fix it subsequently on a piece ofplastic and cover it with transparent foil. Leave it for 30 min.

9. Put X-ray film on the covered membrane and expose it for 30 min.Carry out possibly shorter or longer (maximally for 2 h) exposures.

10. After exposing the film successfully put the membrane again into thedish and strip it with 120 ml of preheated buffer IV (5 mM EDTA/1% ofSDS, 85° C.) 6× for 5 min.

11. Wash the membrane 1× with 120 ml of buffer V (50 mM Tris HCI/150 mMNaCl) and begin again with step 1. If the membrane is not to beimmediately rehybridized it may be stored intermediately in buffer IV orbetween transparent foil.

What is claimed is:
 1. A method for detecting membrane-bound nucleicacids comprising: i) preincubating the membrane in a hybridizationbuffer consisting of 500 ml of 10×TNT, 1250 ml of 20% SDS, 10 g ofblocking reagent and 3250 ml of H₂O; ii) hybridizing the nucleic acidswith a non-radioactively labeled probe in said hybridization buffer;iii) washing at least twice in a buffer I consisting of 10 g of blockingreagent, 250 ml of 10×PBS, 125 ml of 20% SDS and 4625 ml of H₂O; iv)incubating the membrane with a streptavidin alkaline phosphataseconjugate in buffer I; v) washing the membrane once in buffer I, thenwashing at least twice in a buffer II consisting of 250 ml of 10×PBS,125 ml of 20% SDS, 4625 ml of H₂O; then equilibrating the membrane in abuffer III consisting of 64.4 g of Tris HCl, 1770 ml of H₂O, 210.4 g ofdiethanol amine, pH 9.75; vi) transferring the membrane to a solution ofan alkaline phosphatase substrate in said buffer III; and vii) detectinga signal from a reaction of said alkaline phosphatase substrate withsaid alkaline phosphatase conjugate.
 2. The method of claim 1, whereinthe alkaline phosphatase substrate is (disodium2-chloro-5-(4-methoxyspiro{1,2-dioxetane-3,2′-(5-chloro)-tricyclo[3.3.1.1^(3,7)]decan}-4-yl))-1-phenylphosphate.
 3. The method of claim 1, wherein all of steps i) to vii) areconducted at room temperature.
 4. The method of claim 2, wherein all ofsteps i) to vii) are conducted at room temperature.
 5. The method ofclaim 1, 2, 3, or 4 wherein the detecting step vii) is performed usingX-ray film.
 6. A kit comprising, in separate containers: a hybridizationbuffer comprising 10×TNT, 20% SDS, and a blocking reagent; 10×PBScontaining Na₂HPO₄, NaH₂PO₄, and NaCl, pH approximately 7.3; a buffer Icomprising a blocking reagent, 10×PBS and 20% SDS; a buffer IIcomprising 10×PBS and 20% SDS; 10× buffer III comprising Tris-HCl anddiethanol amine at pH 9.75, a buffer IV comprising EDTA and 20% SDS; abuffer V comprising NaCl and Tris-Cl, pH 8.0; and 10×TNT comprisingTris-HCl, Tris-base, NaCl and Triton X-100, pH approximately 8.0.
 7. Akit comprising, in separate containers: a) a hybridization bufferconsisting essentially of 500 ml 10×TNT 1250 ml of 20% SDS 10 g ofblocking reagent 3250 ml of H₂O b) 10×PBS consisting essentially of516.2 g of Na₂HPO₄ 132.6 g of NaH₂PO₄ 198.7 g of NaCl H₂O to make 5 l a)buffer I consisting essentially of 10 g of blocking reagent 250 ml10×PBS 125 l of 20% SDS 4625 ml of H₂O d) buffer II consistingessentially of 250 ml 10×PBS 125 ml of 20% SDS 4625 ml of H₂O e)10×buffer III consisting essentially of 64.4 g of Tris HCl 1770 ml ofH₂O 210.4 g of diethanol amine f) buffer IV consisting essentially of4700 ml of H₂O 50 ml of 0.5 M EDTA, pH 8.0 250 ml of 20% SDS g) buffer Vconsisting essentially of 920 ml of H₂O 30 ml of 5 M NaCl 50 ml 1 M TrisCl, pH 8 h) 10×TNT consisting essentially of 44.4 g of Tris HCl 25.6 gof Tris base 73 g of NaCl 818 ml of H₂O 100 ml of Triton X-100.